Three Component One-Pot Synthesis and Antiproliferative Activity of New [1,2,4]Triazolo[4,3-a]pyrimidines

A series of new [1,2,4]triazolo[4,3-a]pyrimidine derivatives was prepared using a one-pot three-component synthesis from 5-amino-1-phenyl-1H-1,2,4-triazoles, aromatic aldehydes and ethyl acetoacetate. The compound structures were confirmed by IR, 1H-NMR, 13C-NMR, HRMS and X-ray analyses. The biological activity of these compounds as antitumor agents was evaluated. Their antitumor activities against cancer cell lines (MDA-MB-231 and MCF-7) were tested by the MTT in vitro method. Among them, compounds 4c and 4j displayed the best antitumor activity with IC50 values of 17.83 μM and 19.73 μM against MDA-MB-231 and MCF-7 cell lines, respectively, compared to the Cisplatin reference.


Introduction
Heterocyclic compounds have received special attention in organic chemistry due to their presence in many natural products and their diverse biological properties [1]. Among them, pyrimidines and their derivatives occupy an important place due to their biological importance for medicinal chemistry [2][3][4].
A review of the literature also revealed that substituted 1,2,4-triazoles and their bridged heterocyclic derivatives have attracted particular attention over the last two decades due to their wide range of therapeutic properties [5]. Fusion of the 1,2,4-triazole ring with the pyrimidine ring gives rise to the formation of bicyclic heterocycles called 1,2,4triazolopyrimidines (TPs), where the common nitrogen of the triazole and pyrimidine occupy the ring junction. These triazole-fused pyrimidines have been the subject of increasing interest for their various important pharmaceutical properties [6][7][8]. They appear in many synthetic pharmacophores that possess antiparasitic, antimicrobial, anticancer and antibiotic activities [9][10][11]. In particular, many triazolo [4,3-a]pyrimidines exhibited antiproliferative effects against HePG-2 and MCF-72a cell lines [12] and cytotoxic activity on a human hepatic carcinoma cell line (HEPG2) [13]. For example, 1,2,4-triazolo [4,3a]pyrimidines A and B were reported to show antibacterial activity [14,15], and compound B showed higher DNA photocleavage activity [16] and has been described as a potent apoptotic inducer [17] (Figure 1).
In the continuity of the syntheses developed within our group using condensed pyrazoles, which have proven to be of biological interest [28][29][30][31], we report herein the three-component synthesis of [1,2,4]triazolo [4,3-a]pyrimidines in a one-pot manner and preliminary results for in vitro antiproliferative evaluation. Due to their flexibility to assemble several reagents in a short time and transform them into compounds of interest in a one-pot procedure [32,33], multi-component reactions (MCRs) have become very popular in the development of new biologically active compounds. Additionally, MCRs are favored for their experimental simplicity, their atom-/step economy, their reduced reaction time and the high yields produced compared to classical chemistry [34]. Note that syntheses of some condensed triazolopyrimidine derivatives using multicomponent one-pot methods have also been reported [35,36].

Chemistry
In our initial investigation, we sought to optimize the conditions for this multicomponent reaction using aminotriazole 1a, benzaldehyde 2a, and ethyl acetoacetate as starting reagents for the reaction model, by varying the catalyst and the solvent (Table 1).
In our first attempts, we performed the reaction using 10 mol% of paratoluene sulphonic acid (APTS) as a catalyst and H 2 O as a solvent under reflux for 24 h, but only traces of the desired product 4a were observed (Table 1, entry 1). Interestingly, when water was replaced by ethanol at reflux, the desired product 4a was isolated in 75% yield (Table 1, entry 2). Under the same conditions but replacing APTS with HCl, the expected product 4a was formed in a low yield (45%) ( Table 1, entry 3). This decrease in yield was even more pronounced when acetic acid was used as a catalyst (Table 1, entry 4). The use of a basic catalyst such as piperidine proved unsuccessful (Table 1, entry 5). In the absence of any solvent and catalyst, and by heating until the reaction mixture had completely melted, the desired product 4a was isolated with a yield not exceeding 10% (Table 1, entry 7). A reduction in the reaction time to 18 h at the reflux of ethanol in the presence of 10 mol% of APTS resulted in an incomplete conversion rate. The use of a polar aprotic solvent such as acetonitrile did not give complete satisfaction since the yield of the isolated product 4a was only 4 50% (Table 1, entry 6). Consequently, following the results recorded in Table 1, the best conditions for obtaining compound 4a with the best yield were the use of APTS as a catalyst (10 mol%) at the reflux of ethanol for 24 h (Table 1, entry 2).  In our first attempts, we performed the reaction using 10 mol% of paratoluene sulphonic acid (APTS) as a catalyst and H2O as a solvent under reflux for 24 h, but only traces of the desired product 4a were observed ( Table 1, entry 1). Interestingly, when water was replaced by ethanol at reflux, the desired product 4a was isolated in 75% yield (Table 1, entry 2). Under the same conditions but replacing APTS with HCl, the expected product 4a was formed in a low yield (45%) ( Table 1, entry 3). This decrease in yield was even more pronounced when acetic acid was used as a catalyst (Table 1, entry 4). The use of a basic catalyst such as piperidine proved unsuccessful (Table 1, entry 5). In the absence of any solvent and catalyst, and by heating until the reaction mixture had completely melted, the desired product 4a was isolated with a yield not exceeding 10% (Table 1, entry 7). A reduction in the reaction time to 18 h at the reflux of ethanol in the presence of 10 mol% of APTS resulted in an incomplete conversion rate. The use of a polar aprotic solvent such as acetonitrile did not give complete satisfaction since the yield of the isolated product 4a was only 4 50% (Table 1, entry 6). Consequently, following the results recorded in Table 1, the best conditions for obtaining compound 4a with the best yield were the use of APTS as a catalyst (10 mol%) at the reflux of ethanol for 24 h (Table 1, entry 2).
As illustrated in Scheme 1, the three-component reaction is compatible with a wide range of aromatic aldehydes containing electron donating or electron withdrawing substituents. Indeed, aldehydes carrying electron donor groups such as CH 3 , C 2 H 5 or electron withdrawing groups such as Cl and F were easily condensed on 5-aminotriazoles 1a-b in the presence of ethyl acetoacetate, providing access directly to the desired compounds in good to excellent yields. In addition, this reaction was also compatible with aldehydes substituted by a strongly electron-donating group such as OCH 3 or a strongly electronwithdrawing group such as NO 2 . Likewise, the use of an aldehyde substituted by two chlorine atoms on the aromatic ring successfully led to the desired products 4g and 4n with respective yields of 85 and 80%.
As also observed, the efficiency of this multicomponent reaction was not affected by the nature of these substituents. Similarly, there was no significant difference in the yields of the reactions depending on the nature of the substituents, either on the aminotriazole or on the aromatic aldehyde.
Based on the above results and drawing inspiration from the results reported in the literature [37,38], we hypothesized that the mechanism of the three-component one-pot reaction could be as follows (Scheme 2).
The reaction begins with an attack of the pi doublet of the enol form of ethyl acetoacetate 3 on the carbonyl of the aromatic aldehyde leading subsequently, after the elimination of a molecule of water, to the corresponding arylidene according to a known Knoevenageltype reaction. After that, the reaction can proceed along two different pathways: In pathway a, there is a 1,4-Michael-type addition of a doublet of the amino group of aminotriazole 1 on the most electrophilic carbon of the arylidene, leading to a reaction intermediate, which undergoes intramolecular lactamization followed by dehydration to lead to compound 4 .
In pathway b, it is the doublet of nitrogen 4 of 5-amino-1-phenyl-1H-1,2,4-triazole 1 that attacks the electrophilic carbon of the arylidene, which, after intramolecular cyclization followed by dehydration, leads to isomer 4. This regioselectivity of the reaction appears logical insofar as the Michael addition involves the most nucleophilic nitrogen atom of the aminotriazole 1. To elucidate the real structures of the synthesized compounds 4a-n, we carried out a 2D NMR HMBC sequence on compound 4f, since one-dimensional NMR does not make it possible to decide between these two isomers of position 4 and 4. We observed on the one hand a correlation between the C3 carbon and the H5 proton, and on the other hand a correlation between C5 and the methyl group protons attached to the C7 carbon (see Figure S27 in the Supplementary Materials), thus proving the unique formation of isomer 4.
In addition to evidence from 2D NMR spectroscopic analysis, X-ray crystallographic analyses of single crystals of compounds 4a and 4f strongly confirmed the structures of the cyclo-condensation compounds 4a-n (see Figure S2 and the Supplementary Materials for X-ray data of 4a and 4f). The ORTEP diagrams shown in Figure 2 clearly show the formation of type 4 compounds, thus unambiguously confirming the high regioselectivity of this reaction [39]. Next, the biological potential of the synthesized compounds 4a-n as antitumor agents was subsequently evaluated.

Biological Activity: Anticancer Activity In Vitro
All target compounds 4a-n were evaluated for their antitumor activity in vitro by the MTT method. The IC 50 values against MDA-MB-231 and MCF-7 (human breast cancer cell lines) are summarized in Table 2.  Table 2 shows that the synthesized triazolopyrimidines 4a-n exhibited inhibition of the proliferative activity of MDA-MB-231 and MCF-7 cancer cells. These compounds make it possible to improve the efficacy of the antitumor response compared with Cisplatin, particularly as the most potent inhibitory molecules are those that have a more significant decrease in IC 50 values. These values are 17.83 µM and 20.33 µM for compound (4c) bearing R = CH 3 , R 1 = H, R 2 = CH 3 O, and 20.97 µM and 19.73 µM for compound (4j) bearing R = CH 2 -CH 3 , R 1 = H, R 2 = CH 3 O, respectively, P 2 times and 4 times more potent than Cisplatin (one of the most effective FDA-approved treatments for breast cancer [40]). It appears, in this case, that the presence of the CH 3 O moiety in the aryl group increases antitumoral activity.
The most potent compounds (4c and 4j) will be the subject of more biological experiments leading to protein target identification and guided by docking studies in order to determine the mechanisms that induced tumor cell death. An entire investigation of cell death and the cell cycle of treated cells is in progress.

General Information
All thin layer chromatography (TLC) analyses were performed on type 60 F 254 silica gel n silica-gel-precoated aluminum sheets (Type60 F254, 0.25-mm thickness; from Merck, Darmstadt, Germany) with detection using a UV lamp. Melting points were determined on the Kofler and Buchner bench and were not corrected. Infrared (IR) spectra were recorded using a Perkin Elmer device whose range of precision is from 4000 to 400 cm −1 in powders (dispersed in a KBr pellet). All NMR spectra were recorded using a Brucker AV400 Avance spectrometer (at 400 MHz for 1 H and 100 MHz for 13 C). Chemical shifts are expressed in parts per million (ppm) using TMS as an internal standard in CDCl 3 . 5-Amino-1-phenyl-1H-1,2,4-triazole 1a-b was prepared according to the literature [41] by gently refluxing in methanol.
All other reagents and solvents used were analytical grade.

Pharmacology
We adopted the same method as Msalbi et al. [42]. Compounds were solubilized in DMSO and serially diluted with cell culture media just before use.
For MCF-7 and T47D, the cell lines are obtained from the Institut Pasteur in Tunis (Tunisia). For line MDA-MB-231, it is obtained from our colleague Mohamed Abdelkarim, Faculty of Medicine of Tunis. This line was the subject of two publications by our colleague.
Human breast cancer cell lines MDA-MB-231 (ER-, PR-, HER2-) and MCF7 (ER+, PR+, HER2-) were cultured in DMEM medium supplemented with 10% fetal bovine serum, 50 IU/mL penicillin and 50 mg/mL streptomycin and maintained in an incubator at 37 • C in a humidified atmosphere at 5% CO 2 . The MTT test was performed to determine the effect of our chemical compounds on breast cancer cell lines MDA-MB-231 and MCF7 compared to Cisplatin. A 96-well microtiter plate containing 0.1 mL DMEM/well was seeded with 8.103 of each cell line. After 24 h of culture, the cells were treated with these compounds and Cisplatin for 48 h with different concentrations ranging from 0 to 100 µM. After exposure, the medium was removed and MTT solution was added (5 mg/mL) to each well containing 100 µL of DMEM. After 4 h, a formazan precipitate was dissolved in 100 µL/well 10% SDS and recorded at 570 nm. The IC 50 value was defined as cell viability ratio (%) = (OD treated/OD untreated) × 100. All the experiments were carried out in triplicate with six measurements for each concentration tested.

Conclusions
In conclusion, we have developed a simple and efficient strategy for preparation of [1,2,4]triazolo [4,3-a]pyrimidine derivatives through a one-pot three-component reaction from easily available 5-amino-1-phenyl-1H-1,2,4-triazoles, aromatic aldehydes and ethyl acetoacetate using 10 mol% APTS as a catalyst. All synthesized products are new and have been isolated with good yields. The regioselectivity of this developed condensation was confirmed by 2D NMR analysis and X-ray crystallographic analyses. Fourteen of the newly synthesized compounds were screened for their biological activities and found to be more active against the tumor cell lines MDA-MB-321 and MCF-7 compared to Cisplatin. The results showed that among this series, compounds 4c and 4j exhibited the best antitumor activity against breast cancer with respective IC 50 values of 17.83 µM, and 19.73 µM. These will be taken into account in further study.